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United States Patent |
6,091,984
|
Perelman
,   et al.
|
July 18, 2000
|
Measuring tissue morphology
Abstract
The present invention relates to systems and methods for measuring one or
more physical characteristics of material such as tissue using optical
radiation. The system can use light that is scattered by a tissue layer to
determine, for example, the size of nuclei in the tissue layer to aid in
the characterization of the tissue. These methods can include the use of
fiber optic devices to deliver and collect light from a tissue region of
interest to diagnose, for example, whether the tissue is normal or
precancerous.
Inventors:
|
Perelman; Lev T. (Malden, MA);
Backman; Vadim (Cambridge, MA);
Feld; Michael S. (Newton, MA);
Zonios; George (Cambridge, MA);
Itzkan; Irving (Boston, MA);
Manoharan; Ramasamy (Wooster, OH)
|
Assignee:
|
Massachusetts Institute of Technology (Cambridge, MA)
|
Appl. No.:
|
948734 |
Filed:
|
October 10, 1997 |
Current U.S. Class: |
600/476 |
Intern'l Class: |
A61B 005/00 |
Field of Search: |
600/476,478,310
356/432
|
References Cited
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5402778 | Apr., 1995 | Chance | 128/633.
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5419321 | May., 1995 | Evans | 128/633.
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5452723 | Sep., 1995 | Wu et al. | 128/664.
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5460177 | Oct., 1995 | Purdy et al. | 128/633.
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5491344 | Feb., 1996 | Kenny et al. | 250/461.
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5560356 | Oct., 1996 | Peyman | 128/633.
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5582168 | Dec., 1996 | Samuels et al. | 128/633.
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5582169 | Dec., 1996 | Oda et al. | 128/633.
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5596987 | Jan., 1997 | Chance | 128/633.
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5596992 | Jan., 1997 | Haaland et al. | 128/664.
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5625458 | Apr., 1997 | Alfano et al. | 356/446.
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5630423 | May., 1997 | Wang et al. | 128/664.
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5636633 | Jun., 1997 | Messerschmidt et al. | 128/633.
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5640247 | Jun., 1997 | Tsuchiya et al. | 356/446.
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5713364 | Feb., 1998 | DeBaryshe et al. | 128/664.
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5733739 | Mar., 1998 | Zakim et al. | 435/29.
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5813987 | Sep., 1998 | Modell et al. | 600/473.
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5919140 | Jun., 1999 | Perelman et al. | 600/476.
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5931789 | Aug., 1999 | Alfano et al. | 600/473.
|
Foreign Patent Documents |
92/14399 | Sep., 1992 | WO.
| |
96/29926 | Mar., 1996 | WO.
| |
96/28084 | Sep., 1996 | WO.
| |
Other References
Newton R. G., Scattering Theory of Waves and Particles, Second Edition
Chapter 2, "Spherically Symmetric Scatterers," pp. 30-53. Chapter 3,
"Limiting Cases and Approximations," pp. 54-78.
|
Primary Examiner: Lateef; Marvin M.
Assistant Examiner: Mercader; Eleni Mantis
Attorney, Agent or Firm: Hamilton, Brook, Smith & Reynolds, P.C.
Goverment Interests
GOVERNMENT SUPPORT
The invention was supported, in whole or in part, by a Grants No.
P41RR02594 and CA53717 from the National Institutes For Health. The
Government has certain rights in the invention.
Claims
What is claimed is:
1. A method of measuring a size of a structure in a layer of tissue
comprising:
directing incident radiation from a broadband light source onto a region of
interest in the layer of tissue;
collecting scattered radiation from the tissue at a plurality of
wavelengths;
detecting the collected scattered radiation to provide a measured spectrum
as a function of wavelength; and
determining a size of a structure within the tissue layer with the measured
spectrum.
2. The method of claim 1 further comprising determining if the region of
interest includes dysplastic tissue.
3. The method of claim 1 further comprising directing radiation onto the
tissue using a fiber optic probe.
4. The method of claim 1 further comprising collecting the radiation from
the tissue with a fiber optic probe.
5. The method of claim 1 further comprising determining an average nuclear
size of nuclei within the region of interest.
6. The method of claim 1 further comprising measuring a diameter of a
tissue nucleus within the region of interest.
7. The method of claim 1 further comprising measuring a periodic component
of an intensity of radiation from the tissue as a function of wavelength.
8. The method of claim 7 further comprising determining the size of a
nucleus in the tissue from the periodic component.
9. A method of optically measuring tissue comprising:
directing incident radiation onto a region of interest in tissue to be
measured;
collecting scattered radiation from the tissue; and
measuring a periodic component of collected scattered radiation as a
function of wavelength to determine a physical characteristic of the
tissue.
10. The method of claim 9 further comprising determining if the region of
interest includes dysplastic tissue.
11. The method of claim 9 further comprising directing radiation onto the
tissue using a fiber optic probe.
12. The method of claim 9 further comprising collecting the radiation from
the tissue with a fiber optic probe.
13. The method of claim 9 further comprising determining an average nuclear
size of nuclei within the region of interest.
14. The method of claim 9 further comprising measuring a diameter of a
tissue nucleus within the region of interest.
15. The method of claim 9 further comprising collecting radiation with an
endoscope, the endoscope having an imaging sensor at a distal end of the
endoscope.
16. The method of claim 9 further comprising determining a density of
nuclei in the tissue from the periodic component.
17. A method of determining a presence of dysplasia in tissue comprising:
directing incident radiation onto a region of interest in an epithelial
layer of tissue;
collecting backscattered radiation from the tissue;
detecting the collected backscattered radiation with a detector;
determining a size of a structure within the epithelial layer of tissue
using the detected radiation; and
determining the presence of dysplasia in the region of interest of the
tissue.
18. The method of claim 17 further comprising determining if the region of
interest includes dysplastic tissue.
19. The method of claim 17 further comprising collecting radiation in the
range of 350 nm to 700 nm.
20. The method of claim 17 further comprising collecting the radiation from
the tissue with a fiber optic probe.
21. The method of claim 17 further comprising determining an average
nuclear size of nuclei within the region of interest.
22. The method of claim 17 further comprising measuring a diameter of a
tissue nucleus within the region of interest.
23. The method of claim 17 further comprising measuring a periodic
component of an intensity of scattered radiation from the tissue as a
function of wavelength.
24. The method of claim 17 further comprising determining the size of a
nucleus in the tissue from the periodic component.
25. A method of optically measuring a material comprising:
directing incident radiation onto a region of interest in the material
tissue to be measured;
collecting scattered radiation from the material;
detecting a scattering spectrum from the collected scattered radiation; and
measuring a periodic component of collected scattered radiation as a
function of wavelength to determine a physical characteristic of the
material.
26. The method of claim 25 further comprising directing radiation onto the
material using a fiber optic probe.
27. The method of claim 25 further comprising collecting the radiation from
the material with a fiber optic probe, the probe having an optical fiber
with a numerical aperture in a range of 0.05-0.25.
28. The method of claim 25 further comprising determining an average
nuclear size of nuclei within the region of interest.
29. The method of claim 25 further comprising measuring a number of
particles per unit area within the region of interest.
30. An apparatus for optically measuring tissue comprising:
a radiation source that illuminates a region of interest in tissue to be
measured with incident radiation;
an optical system that collects scattered radiation from the tissue;
a detector system that senses the collected scattered radiation; and
a data processor that determines a periodic component of detected radiation
as a function of wavelength to determine a physical characteristic of the
tissue.
31. The apparatus of claim 30 further comprising a broadband light source
that generates light in a range of 350-700 nm.
32. The apparatus of claim 30 further comprising a fiber optic probe that
couples the source to the tissue.
33. The apparatus of claim 30 further comprising a fiber optic probe that
collects the light in a collection angle between 2 and 12 degrees.
34. The apparatus of claim 33 wherein the probe is insertable in an
endoscope.
35. A method of measuring a size of a structure in a layer of tissue
comprising:
directing incident radiation onto a region of interest in the layer of
tissue;
collecting scattered radiation from the tissue;
detecting the collected scattered radiation; and
determining an average nuclear size of nuclei within the region of interest
using the detected radiation.
36. The method of claim 35 further comprising determining if the region of
interest includes dysplastic tissue.
37. The method of claim 35 further comprising directing radiation onto the
tissue using a fiber optic probe.
38. The method of claim 35 further comprising collecting the radiation from
the tissue with a fiber optic probe.
39. The method of claim 35 further comprising measuring a diameter of a
tissue nucleus within the region of interest.
40. The method of claim 35 further comprising measuring a periodic
component of an intensity of radiation from the tissue as a function of
wavelength.
41. The method of claim 40 further comprising determining the size of a
nucleus in the tissue from the periodic component.
42. A method of measuring a size of a structure in a layer of tissue
comprising:
directing incident radiation onto a region of interest in the layer of
tissue;
collecting scattered radiation from the tissue;
detecting the collected scattered radiation; and
measuring a diameter of a tissue nucleus within the region of interest with
the detected radiation.
43. The method of claim 42 further comprising determining if the region of
interest includes dysplastic tissue.
44. The method of claim 42 further comprising directing radiation onto the
tissue using a fiber optic probe.
45. The method of claim 42 further comprising collecting the radiation from
the tissue with a fiber optic probe.
46. The method of claim 42 further comprising determining an average
nuclear size of nuclei within the region of interest.
47. The method of claim 42 further comprising measuring a periodic
component of an intensity of radiation from the tissue as a function of
wavelength.
48. The method of claim 47 further comprising determining the size of a
nucleus in the tissue from the periodic component.
Description
BACKGROUND OF THE INVENTION
Methods for diagnosis of cancer at an early stage are essential for cancer
prevention and therapy. Many types of cancers grow from epithelial
tissues, which cover inner and outer surfaces of the human body. Many of
these, for example cancer in gastrointestinal tract, progress through the
stage of dysplasia. Dysplasia can be defined as neoplastic tissue which is
not malignant yet, but is considered to be a precursor of malignancy. If
diagnosed at this stage, most tumors are curable. In the case of
gastrointestinal tumors, current methods of diagnosis are based on
endoscopy. However, dysplastic tissue is frequently not endoscopically
apparent. Thus, detection of dysplasia in the gastrointestinal tract and
other sites often relies on sporadic sampling for this "invisible"
malignant precursor. However, sporadic biopsies have a high probability of
missing dysplastic changes. In some cases the biopsy procedure is
impossible.
Efforts toward a substitution for standard biopsies have been made in order
to provide accurate diagnosis of cancerous tissue in different organs in
vivo and in real time. For this purpose, optical techniques that are
non-invasive do not require tissue removal and can be performed in-vivo.
Such methods provide information at the microscopic level and can thus
provide for the search for very small sites which are likely to be missed
by standard biopsies. While most human organs can be diagnosed by means of
optical techniques, they are particularly applicable to the tissues in
human body lumens, since they are easily accessible by optical probes,
which can be inserted into one of the channels of a conventional
endoscopic tube.
SUMMARY OF THE INVENTION
The present invention relates to the use of light to determine physical
characteristics of a structured layer of material, and in particular
certain qualitative information regarding the morphology of tissue
structures using scattered light. Both backscattered and transillumination
methods can be used, depending upon the thickness of the material and the
size and distribution of the structure being measured. Examples of
properties of materials that can be measured include surface roughness,
parasity, cytometer measurements, or any material in which changes in the
refractive index of a material correspond to changes in structures. This
type of scattering spectroscopy can be differentiated from absorption
spectroscopy which is unable to quantitatively measure particle
morphology.
Despite extensive investigations, no reliable optical technique to diagnose
dysplasia in-vivo is known. One of the difficulties resides in the fact
that dysplastic changes are limited to the uppermost epithelial layer,
which can be as thin as 20 .mu.m, a one cell layer that is nearly
transparent to optical radiation.
Tissue in the gastrointestinal tract, for example (other hollow organs
share the same features also), is covered by a layer of cells called
epithelium (from 20 .mu.m to 300 pm thick depending on the part of the
tract) supported by relatively acellular and highly vascular loose
connective tissue, lamina propria, which can be up to 500 .mu.m in
thickness and contains a network of collagen and elastic fibers, and
variety of white blood cell types. Beneath the lamina propria there is a
muscular layer, muscularis mucosae, (up to 400 .mu.m thick) and another
layer of moderately dense connective tissue called submucosa (400-600
.mu.m thick) containing many small blood vessels and abundant collagen and
elastic fibers. The overall thickness of those layers is about 1 mm. Since
a characteristic penetration depth of optical radiation into biological
tissue does not usually exceed 1 mm, for a preferred embodiment it is
sufficient to limit measurements of tissue by those layers.
Adenocarcinoma of the esophagus arises in metaplastic columnar epithelial
cells in the esophagus, termed "Barrett's esophagus", which is a
complication of long-standing gastrointestinal reflux. In this condition,
the distal squamous epithelium is replaced by columnar epithelium
consisting of a one cell layer which resembles that found in the
intestines. Barrett's esophagus is frequently associated with dysplasia
which later can progress to cancer. Trials of endoscopic surveillance of
patients with Barrett's esophagus have not resulted in a reduction of
esophageal cancer mortality. The most likely explanation is that dysplasia
occurring in the esophagus cannot be seen with standard endoscopic imaging
and sporadic biopsy sampling is necessary. This procedure can sample only
about 0.3% of the tissue at risk. Thus, there is tremendous potential for
sampling error.
The application of optical techniques to diagnose dysplasia in Barrett's
esophagus is limited by the fact that the primary alterations in the
tissue occur in the epithelium which is one cell thick (.about.20-30
.mu.m) while fluorescence or reflectance spectra are mostly formed in
deeper tissue layers. One of the most prominent features of a dysplastic
epithelium is the presence of enlarged, hyperchromatic, and crowded
nuclei. In fact, these changes in nuclei size and spatial distribution are
the main markers used by a pathologist to diagnose a tissue specimen as
being dysplastic. No significant changes in other tissue layers is
observed. Unfortunately, epithelium does not contain strong absorbers or
fluorophores, and the thickness of the epithelium is relatively small and
thus negligible. These make epithelium diagnosis in Barrett's esophagus to
be a difficult problem.
A preferred embodiment of the present invention relates to a system of
measuring a fine structure component in backscattered light from mucosal
tissue which is periodic in wavelength. This structure is ordinarily
masked by a diffusive background, which must be removed to render it
observable. The origin of this component is due to light which is
Mie-scattered by surface epithelial cell nuclei. By analyzing the
amplitude and frequency of the periodic structure, the density and size
distribution of these nuclei can be extracted. These quantities are
important indicators of neoplastic precancerous changes in biological
tissue, and the technique can thus provide a useful tool for observing
such changes in patients undergoing endoscopy.
The light that is incident on the thin layer at the tissue surface is not
completely randomized. In this thin region the details of the elastic
scattering process can be preserved. Mucosal tissues, which line the
hollow organs of the body, generally consist of a thin surface layer of
epithelial cells supported by underlying, relatively acellular connective
tissue. In healthy tissues the epithelium often consists of a single,
well-organized layer of cells with an endface diameter of about 10-20
.mu.m and a height of about 25 .mu.m. In cancerous and pre-cancerous
(dysplastic) epithelium cells proliferate, the cellular layer often
thickens and becomes more tightly packed, and the cell nuclei enlarge and
appear darker (hyperchromatic) when stained. This may indicate increased
nucleic acid density, hence increased refractive index.
A preferred embodiment of the invention utilizes a broadband light source
to illuminate the region of interest in the tissue with optical radiation
in the range between 350 and 700 nm. A fiber optic probe can be used to
deliver and/or collect radiation from the tissue. The system can be used
during endoscopy of a patient to optically survey a body lumen within the
patient and thereby eliminate the need for removal of tissue for biopsy,
or alternatively, can be used to aid in locating tissue suitable for
biopsy.
Backscattered light is preferably collected over a small collection angle
of between 2.degree. and 12.degree., preferably in the range between
3.degree. and 8.degree.. When using an optical fiber system to collect the
scattered light fibers having a numerical aperture between 0.05 and 0.22,
and preferably between 0.07 and 0.1 can be used. Collection angles within
this range reduce the level of background light without loss of the
periodic component in the returning light.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of a fiber optic probe in accordance with the
invention.
FIG. 2 is an enlarged view of the distal end of an endoscope in accordance
with the invention.
FIGS. 3A, 3B and 3C illustrate reflectance spectra from cell monolayers for
normal colon cells (R=0.46); T84 cells (R=0.38); (c) BaSO.sub.4 diffusing
plate (R=1.0)
FIG. 4 illustrates nuclear size distributions from data of FIGS. 3A and 3B,
respectively for normal colon cells; and T84 cells respectively. In each
case, the solid line is the distribution extracted from the data, and the
dashed line is the distribution measured using light microscopy.
FIGS. 5A, 5B and 5C are reflectance spectra from Barretts' esophagus for
diffuse reflectance from a normal site (solid line), a dysplastic site
(dashed line), and the model fit (thick solid line); for corresponding
fine structures; and of resulting nuclear size distributions,
respectively.
FIG. 6 graphically illustrates a comparison of samples analyzed by standard
pathology and the optical methods in accordance with the invention.
FIG. 7 is a system used for in vitro tissue analysis in accordance with the
invention.
FIG. 8 is a process flow diagram illustrating a method of performing an
optical diagnosis of tissue in accordance with the invention.
The foregoing and other objects, features and advantages of the invention
will be apparent from the following more particular description of
preferred embodiments thereof, as illustrated in the accompanying drawings
in which like reference characters refer to the same parts throughout the
different views. The drawings are not necessarily to scale, emphasis
instead being placed upon illustrating the principles of the invention.
DETAILED DESCRIPTION OF THE INVENTION
A preferred embodiment of the invention involves the use of a fiber optic
system to deliver and collect light from a region of interest to measure
one or more physical characteristics of a surface layer. Such a system is
illustrated in FIG. 1. This system can include a light source 50 such as a
broadband source, a fiber optic device 10 for delivery and/or collection
of light from the tissue, a detector system 80 that detects the scattered
light from the tissue, a computer 120 having a memory 122 that analyzes
and stores the detected spectra, and a display 124 that displays the
results of the measurement. A lens 60 can be used to couple light from the
source 50 into the excitation fiber 30 of the probe 10. A filter 110 and
lens system 90,100 can be used to efficiently couple collected light to a
spectrograph 70. A controller 140 connected to the data processing system
120 can be connected to a clock and a pulser 150 that controls the light
source 50.
The distal end 15 of the probe 10 is illustrated in FIG. 2 where the
central excitation fiber 30 is surrounded by six peripheral collection
fiber 20. The distal end of the device can be enclosed in an optical
shield 25 such as that described in U.S. Pat. No. 5,199,431, the entire
contents of which is incorporated herein by reference. Other endoscopic
devices can be used such as an optical needle as described in the above
referenced patent or as described in U.S. Pat. No. 5,280,788, the entire
contents of which is also incorporated herein by reference.
The collection fibers 20 preferably have a numerical aperture in the range
of 0.05 to 0.22 in order to provide a desired collection angle from the
material being measured. This aids in reducing background that is removed
from scattering spectrum without loss of the periodic component.
The collection fibers can also be replaced or supplemented by a distally
mounted imaging sensor 35 such as a charged coupled device or CMOS imager.
The sensor has a pixellated structure that is sensitive to the different
colors contained in the scattering spectrum being recorded. Further
details regarding the use of a distally mounted sensor can be found in
U.S. Ser. No. 08/745,509 filed on Nov. 12, 1996, the entire contents of
which is incorporated herein by reference.
The backscattered light collected with this system can be analyzed to
determine certain physical characteristics of epithelial tissue. The
relationship between the collected light and the physical characteristics
to be determined using this light can be described as follows.
Epithelial nuclei can be represented as spheroidal Mie scatterers with a
refractive index higher than that of the surrounding cytoplasm. Normal
nuclei have a characteristic diameter l=4-7 .mu.m. In contrast, dysplastic
nuclei can be as large as 20 .mu.m in size, occupying almost the entire
cell volume. Thus, in the visible range, the wavelength .lambda.<<l, and
the component of light scattered by the nuclei will exhibit a periodicity
with wavelength, the details of which are determined by the nuclear size
distribution. The Van de Hulst approximation can be used to describe the
optical scattering cross section of the nuclei:
##EQU1##
where .delta.=.pi.ln.sub.c (n-1), with n.sub.c the refractive index of
cytoplasm and n the refractive index of the nuclei relative to that of
cytoplasm.
When a beam of light is incident on an epithelial layer of tissue, a
portion of this light is backscattered from the epithelial nuclei, while
the remainder is transmitted to deeper tissue layers, where it undergoes
multiple scattering and becomes randomized. All of the diffusive light
which is not absorbed in the tissue eventually returns to the surface,
passing once more through the epithelium, where it is again subject to
scattering from the cell nuclei. Thus, the emerging light consists of a
large diffusive background plus the component of forward scattered and
backscattered light from the nuclei in the epithelial layer. For a thin
slab of epithelial tissue containing nuclei with size distribution N(l)
(number of nuclei per unit area (mm.sup.2) and per unit interval of
nuclear diameter (.mu.m)), the approximate solution of the transport
equation for the reflectance R(.lambda.) collected by an optical probe
with acceptance solid angle .OMEGA..sub.c is given by the following
expression:
##EQU2##
where I.sub.i ((.lambda.,s)) is the intensity of the incident light
delivered in solid angle .OMEGA..sub.i,I.sub.d (.lambda.,s) is the
intensity of the light emerging from the underlying tissue, and
<I(s)>.OMEGA.=.intg..sub..OMEGA. I(s)ds for any function I(s) and solid
angel .OMEGA., with s a unit vector pointing outward from the tissue
surface in an arbitrary direction. The quantity R(.lambda.)=<I.sub.d
(.lambda.,s)>.OMEGA..sub.c /<I.sub.i (.lambda.,s)>.OMEGA..sub.c is the
reflectance of the diffusive background. The optical distance
##EQU3##
and scattering phase function
##EQU4##
for a sphere, p(.lambda.,l,s,s.sup.1) is determined by Mie theory. The
first term in Eq. (2) describes the attenuation of the diffusive
background, and the terms in brackets describe backscattering of the
incident light and forward scattering of diffusive background by the
epithelial cell nuclei, respectively.
For small .OMEGA..sub.c the forward scattering term in Eq. (2) can be
expanded in .tau.(.lambda.). Thus,
<(I.sub.d (.lambda.,s.sup.1)p(.lambda.,s,s.sup.1)>2.pi.>.OMEGA..sub.c
/<I.sub.d (.lambda.,s)>.OMEGA..sub.c .congruent.f.sub.0 +f.sub.1
.tau./.tau..sub.0, with
##EQU5##
It is found numerically that f.sub.1 <<f.sub.0 and that f.sub.0 and
f.sub.1 are approximately independent of wavelength in the range of
interest (.lambda..sub.min =360 to .lambda..sub.max =685 nm). Similarly,
for the backscattering term, <(I.sub.i
(.lambda.,-s.sup.1)p(.lambda.,s)>.OMEGA..sub.c /<I.sub.d
(.lambda.,s)>.OMEGA..sub.c .congruent.b.sub.0 -b.sub.1 .tau./.tau..sub.0.
Note that in the forward scattering contribution the first order term
oscillates in phase with .tau.(.lambda.), as required by the optical
theorem, whereas for the backscattering contribution it is out of phase.
Thus, Eq. (2) reduces to
##EQU6##
which shows that the epithelial nuclei introduce a periodic fine structure
component into the reflectance with a wavelength dependence similar to
that of the corresponding scattering cross section. Its periodicity is
approximately proportional to nuclear diameter, and its amplitude is a
function of the size and number of nuclei in the epithelial layer. These
quantities can be determined by analyzing the reflectance, R(.lambda.).
As example of the effects described by Eq. (2), elastic light scattering
from normal T84 tumor human colonic cell monolayers (10 and 15 sites
respectively) was measured and analyzed. The cells, approximately 15 .mu.m
long, were affixed to glass slides in buffer solution and placed on top of
a BaSO.sub.4 diffusing (and highly reflective) plate. The BaSO.sub.4 plate
was used to approximate the diffuse reflectance from underlying tissue.
The diameters of the normal cell nuclei generally ranged from 5 to 7 .mu.m
and those of the tumor cells from 7 to 16 .mu.m.
An optical fiber probe was used to deliver white light from a xenon arc
flashlamp to the samples and collect the return reflectance signal, as
shown in FIG. 1. The probe tip, 1 mm in diameter, consisted of a central
delivery fiber surrounded by six collection fibers, all of which were
covered with a 1 mm thick quartz optical shield. The fibers were 200 .mu.m
core fused silica, NA=0.22 (.OMEGA..sub.i =.OMEGA..sub.c =.pi.NA.sup.2) To
eliminate specular reflection, the probe was beveled at 17.degree. to the
normal. At the proximal end the collection fibers were arranged in a line
and imaged onto the input slit of a spectrograph. A diode array detector
recorded the reflectance spectra from 360 to 685 nm.
FIGS. 3A and 3B show the normalized reflectance R(.lambda.)/R(.lambda.)
from normal and T84 tumor cell samples, respectively. Distinct spectral
features are apparent. For comparison, the reflectance spectrum from the
BaSO.sub.4 plate by itself is also shown in FIG. 3C. This spectrum lacks
structure and shows no prominent features.
To obtain information about the nuclear size distribution from the
reflectance data, Eq. (3) needs to be inverted. The nuclear size
distribution, N(l), can then be obtained from the Fourier transform of the
periodic component of the optical distance .tau.-.tau..sub.0
.congruent.(1-R(.lambda.))/q. The parameter q=1-b.sub.0 -f.sub.0
+2(b.sub.1 -f.sub.1) is associated with forward and backward scattering,
and depends on the probe geometry and the angular distribution of the
incident and reflected light. In this particular example q.apprxeq.0.15.
By introducing the effective wavenumber k=2.PI.n.sub.c
(n-1)/.lambda.-k.sub.0, and k=2.pi.n.sub.c (n-1)/.lambda..sub.max,
K=2.pi.n.sub.c (n-1)
##EQU7##
and we obtain,
##EQU8##
Equation (4) was used to analyze the data. In order to remove spurious
oscillations, N(l) was further processed by convolving it with a Gaussian
filtering function. The solid curves in FIG. 4 show the resulting nuclear
size distributions of the normal and T84 cell monolayer samples extracted
from the spectra of FIGS. 3A and 3B. A nucleus-to-cytoplasm relative
refractive index of n=1.06 and cytoplasm refractive index of n.sub.c =1.36
were used. The dashed curves show the corresponding size distributions,
measured morphometrically via light microscopy. The size distributions can
be approximated by Gaussian distributions. The parameters for those are
presented in Table 1. The extracted and measured distributions are in good
agreement for both normal and T84 cell samples.
TABLE 1
______________________________________
Normal Cells Tumor T84 Cells
Mean Standard
Diameter Standard Mean Deviation
(.mu.m) Deviation (.mu.m)
Diameter (.mu.m)
(.mu.m)
______________________________________
Microscopy
.about.6 .about.0.5 10.2 2.0
Spectroscopy
6.2 0.45 10.1 2.2
______________________________________
The periodic fine structure in diffuse reflectance of esophagus and colon
mucosa of human subjects can be measured during gastroenterological
endoscopy procedures. In the case of Barretts' esophagus, in which the
epithelium consists of a thin monolayer of columnar cells similar to those
used in the cell culture experiments, data were collected as in the cell
culture studies. The optical fiber probe is inserted into the biopsy
channel of the endoscope and brought into contact with the tissue surface.
The methods described herein can also be used to measure structural
properties of other GI tissue, tissues in the oral cavity, the cervix, the
bladder, and skin.
The fine structure component, which is the scattering signature of the cell
nuclei, is typically less than 5% of the total signal and is ordinarily
masked by the background of diffusely scattered light from underlying
tissue, which itself exhibits spectral features due to absorption and
scattering, as shown in FIG. 5A. Its spectral features are dominated by
the characteristic absorption bands of hemoglobin and collagen scattering.
In order to observe the fine structure, this background must be removed.
The absorption length, .mu..sub.a.sup.-1, ranges from 0.5 to 250 mm as the
wavelength is varied, and the effective scattering length (.mu..sub.s
').sup.-1 ranges from 0.1 to 1 mm. Thus, both scattering and absorption
have to be taken into account in subtracting or removing the background
signal.
To represent the background light incident on the tissue is assumed to be
exponentially attenuated, and that at any given depth, z, an amount of
light proportional to the reduced scattering coefficient .mu..sub.s ' is
scattered back towards the surface and further exponentially attenuated.
Since light attenuation depends on both scattering and absorption, the
attenuation coefficient is assumed to be the sum of absorption coefficient
.mu..sub.a and effective scattering coefficient .mu..sub.s.sup.(e)
=.beta..mu..sub.s '. The parameter .beta. was determined by comparison
with Monte Carlo simulations and more accurate models of light transport,
and was found to be .beta..congruent.0.07. Since light only penetrates
.about.1 mm into the tissue, most of the diffusely scattered return light
is confined to the mucosal layer.
The tissue is thereby represented as a two layer medium and neglected
diffusely reflected light from the lower layer. The following approximate
expression for the diffusive light from underlying tissue impinging on the
epithelial cell layer is then obtained:
##EQU9##
with F(s) being a Lambertian function describing the angular dependence of
light emerging from mucosal layer, L a parameter representing the
thickness of the mucosal layer, and L a parameter representing the
thickness of the mucosal layer, and c, the concentration of hemoglobin,
which we find to be the main absorber relative to that of collagen, which
is responsible for light scattering. Because both oxygenated and
deoxygenated hemoglobin are present, the total hemoglobin absorption is
represented as .mu..sub.a =(1-a).mu..sub.a.sup.(Hb)
+.alpha..mu..sub.a.sup.(Hb).sbsp.2.sup.) with oxygen saturation parameter
.alpha.(0.ltoreq..alpha..ltoreq.1).
FIG. 5A shows the reflectance spectra from two Barretts' esophagus tissue
sites, both independently diagnosed by standard pathological analysis to
indicate (1) normal and (2) precancerous (i.e. low grade dysplasia). As
can be seen, the differences in these unprocessed spectra are small. To
analyze them, Eq.(5) was first fit to the broad features of the data by
varying the parameters c, a and L. As seen in FIG. 5A, the resulting fits
are quite accurate. After removing this diffuse background structure by
calculating R(.lambda.)/R(.lambda.), the periodic fine structure is seen
in FIG. 5B. Note that the fine structure from the dysplastic tissue site
exhibits higher frequency content than that from the normal site. Equation
(4) was then employed to extract the respective nuclear size
distributions, yielding FIG. 5C. The difference between normal and
dysplastic tissue sites is evident. The distribution of nuclei from the
dysplastic site is much broader than that from the normal site and the
peak diameter is shifted from .about.7 .mu.m to about .about.10 .mu.m. In
addition, both the relative number of large nuclei (>10 .mu.m) and the
total number of nuclei are significantly increased.
Based on computer analysis, the uncertainty of the above method in
extracting nuclear size information is estimated to be from 5% to 30%,
depending on the noise level and accuracy of the model. The distributions
were calculated using the same refractive index for both normal and
dysplastic nuclei. This is not entirely correct, inasmuch as in stained
histological sections dysplastic nuclei appear hyperchromatic, which may
be indicative of an increase in refractive index. Thus, the relative
number of large nuclei in the distributions measured from dysplastic sites
may be slightly overestimated.
The ability to measure nuclear size distribution in vivo has valuable
applications in clinical medicine. Enlarged nuclei are primary indicators
of cancer, dysplasia and cell regeneration. In addition, measurement of
nuclei of different size can provide information about the presence of
particular cells, and can thus serve, for example, as an indicator of
inflammatory response of biological tissue. This suggests that different
morphology/pathology in the mucosal layer gives rise to distinct patterns
of nuclear distributions.
The physical characteristics that have been found to be useful to
differentiate between Barrett's non-dysplastic and dysplastic epithelium
were the total number of nuclei and the percentage of large nuclei (l>10
.mu.m). A comparison of pathological analysis of samples with the optical
analysis thereof provided the plot (total number of nuclei vs. percentage
of nuclei with a diameter large that 10 .mu.m) in FIG. 6. From those 50
sites, the cumulative sensitivity and specificity, for this analysis were
83% and 100% respectively. The study had a positive predictive value is
100%. The points indicated by n's were either normal or inflamed and those
indicated by d's were displastic. A percentage in the range of 20-30% was
found to be an accurate diagnostic for this type, with 25% being used in
this particular example.
A preferred embodiment of a spectrograph system 300 employed for the
collection of backscattered spectral data from excised tissue samples
using a spectrograph and a charge coupled device (CCD), CMOS or other
integrated solid state imaging array is illustrated in FIG. 7.
System 300 can use a broadband light source or tunable laser 314 for
irradiating a sample 46. Source 314 generates a beam 316 which is directed
by mirror 318 through focusing optics 320 to impinge on sample 46 mounted
on a scattering substrate 325 and behind a transparent window 321. The
beam was focused on the sample at an angle of incidence. The collection
angle 330 can be determined by an aperture or collimator 340 and is
between 2 and 12 degrees, preferably between 3 and 8 degrees.
A portion of the scattered light 322 emitted by sample 46 was collected by
collecting optics 324 a small angle relative to the incident light. In
another preferred embodiment the angle of incidence and collection can be
along a single common axis. Collecting optics 324 collimates and F/matches
the collected light for the spectrograph 310. Prior to entering the
entrance slit of the spectrograph 310, the collected light was passed
through a series of filters 326 which attenuated the unwanted scattered
component of the collected light.
FIG. 8 illustrates generally a process 400 for collecting and analyzing the
scattering spectrum from a material of interest such as tissue. The method
can be performed both in vitro using a microscopy system or a color
imaging system as shown in FIG. 7, or in vivo on a patient. The
illuminating light 402 from a source can use radiation in the range of 300
nm-1200 nm, including the infrared range. After collecting 404 and
detecting 406 radiation, the diffuse background 408 can be removed and the
desired characteristics calculated 410. These results can be used to
provide a diagnosis of the region of interest.
EQUIVALENTS
While this invention has been particularly shown and described with
references to preferred embodiments thereof, it will be understood by
those skilled in the art that various changes in form and details may be
made therein without departing from the spirit and scope of the invention
as defined by the appended claims. Those skilled in the art will recognize
or be able to ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be encompassed in
the scope of the claims.
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